(1) Field of the Invention
The present invention relates to a stable bilayer or multilayer membrane with channels through the membrane and containing a phospholipid and a mixture of a polyhydroxybutyrate (PHB) and a polyphosphate. In particular, the present invention relates to a method for transporting an ion or molecule through the channel in the membrane. Further, the present invention relates to a method for assaying for compounds which block the channel. Finally, the present invention relates to a method for incorporating the channels in membranes.
(2) Description of Related Art
R-poly-3-hydroxybutyrates and inorganic polyphosphates (polypi) are ancient and ubiquitous homopolymers whose biological roles are not well understood. PHB, a head-to-tail polymer of R-3-hydroxybutyrate, is best known as a high molecular weight (60,000 to 1,000,000) polymer deposited within inclusion bodies in many prokaryotes. PolyPi are linear chains of orthophosphate joined by phospho-anhydride bonds, which have a free energy of hydrolysis comparable to that of ATP. Reusch et al (R. N. Reusch and H. L. Sadoff. J. Bacteriol. 156, 778-788 (1983); R. N. Reusch, et al., J. Bacteriol. 168, 553-562 (1986); and R. N. Reusch. Soc. Exp. Biol. and Med. 19, 377-381 (1989)) isolated PHB having a lower molecular weight ( less than 12,000) from bacterial plasma membranes, and from membranes and organelles of plants, and concluded that membrane PHB in Escherichia coli, Azotobacter vinelandii and Bacillus subtilis was complexed with Ca(polyPi). The presence of these complexes in bacterial membranes is discerned by observing the thermotropic fluorescence of the membrane probe, N-phenyl-1-naphthylamine; dissociation of the complexes gives rise to an increase in fluorescence with a peak at ca 56xc2x0 C. The concentration of PHB/polyPi is low during log-phase growth, but increases fifty to a hundred fold or more when the cells are made genetically competent whether by physiological or physico-chemical means. At high concentrations, the complexes cause alterations of the plasma membrane structure observable by freeze-fracture electron microscopy (R. Reusch, et al., Can. J. Microbiol. 33, 435-444 (1987)).
Poly-xcex2-hydroxybutyrate (PHB) and calcium polyphosphate complex membranes were extracted as biological complexes from bacterial membranes (Reusch, R., and Sadoff, H., Proc. National Acad. Science 85, 4176-4180 (1988)). Attempts to reconstitute the complex membranes from calcium polyphosphate and PHB in liposomes met with limited success, since they were significantly disassociated as can be seen from FIG. 1 of this reference. The putative functions of the biological complexes are further discussed in FEMS Microbiology Rev. 103, 119-130 (1992).
Reusch and Sadoff proposed a structure for the PHB/Ca(polyPi) in E. coli, based on molecular and computer modeling with regard for the physical properties of the polymers, the coordination geometry of calcium, and the membrane environment. It assumes that the amphophilic PHB forms a helical porexe2x80x94with a lipophilic exterior of methyl and methylene groups and a hydrophilic lining of ester carbonyl oxygensxe2x80x94that is traversed by the more rigid polypi anion. A channel is formed in the space between the two polymers which has solvating carbonyl oxygens column evenly spaced along its outer wall and negatively-charged binding sites at regular intervals along its inner wall. The channel is subdivided into several contiguous parallel lanes through which cations may move in single-file in the direction of concentration or voltage gradients. Since all cation binding sites are identical, the potential energy minima are also identical. This model of a multiple-site, single-file channel is consistent with current views on protein Ca2+ channel structure expressed by Hess and Tien and Almers and McCleskey (Hess, P. and R. W. Tsien. Nature. 309, 453 (1984); and Almers, W. and McCleskey, E. W., J. Physiol. 353, 585 (1984)). The model is used as a basis for explaining how the inventors believe the present invention functions; however, they do not want to be bound by any particular theory.
It is therefore an object of the present invention to provide ion channel complexes in lipid bilayers or multilayers. It is further an object of the present invention to provide a method for forming ion channel complexes in bilayers which is simple and economical. Further still, it is an object of the present invention to provide an assay method for determining whether the channels are blocked by particular ions or molecules. These and other objects will become increasingly apparent by reference to the following description.
The present invention relates to a stable bilayer or multilayer membrane which has a channel between a first side and a second side of the membrane which comprises: a bilayer (or bilayers) which separates two aqueous regions on each of the sides of the membrane; and a substantially pure mixture of (1) a polyhydroxybutyrate (PHB) and (2) a polyphosphate, the PHB and the polyphosphate having molecular weights which provide a channel across the membrane.
Further the present invention relates to a method for transporting a cation through a channel which comprises: providing a stable bilayer or multilayer membrane which has a channel between a first side and a second side of the membrane which comprises: a lipid bilayer which separates two aqueous regions on each of the sides of the membrane; and a substantially pure mixture of (1) a polyhydroxybutyrate (PHB) and (2) a polyphosphate, the PHB and the polyphosphate having molecular weights which provide a channel across the membrane; and providing transport means for the cation through the channel.
Further still, the present invention relates to a method for assaying a calcium channel blocking compound which comprises: providing a stable bilayer or multilayer membrane which has a channel between a first side and a second side of the membrane which comprises: a lipid bilayer which separates two aqueous regions on each of the sides of the membrane; and a mixture of (1) a substantially pure polyhydroxybutyrate (PHB); and (2) a polyphosphate, the PHB and the polyphosphate having molecular weights which provide a channel across the membrane; providing the calcium channel blocker compound and calcium ions on one or both sides of the membrane; and providing transport means for the calcium ion through the channel, wherein the calcium channel blocking compound blocks the channel through the membrane.
Finally, the present invention relates to a method for forming a bilayer or multilayer membrane which has a channel between a first side and a second side of the membrane which comprises: mixing a phospholipid with a mixture of an inorganic polyphosphate and a polyhydroxybutyrate (PHB) in an organic solvent to provide a membrane forming solution; and forming a membrane between two aqueous phases, wherein the PHB and inorganic polyphosphate form a channel through a bilayer formed by the phospholipid.
The PHB can be economically extracted, sonicated and purified from prokaryotes to produce a molecular weight between 1,000 to 30,000, preferably 11,000 to 16,000. PHB also occurs in higher organisms; however extraction is more difficult. The PHB can also be chemically synthesized by polymerization using well known processes.
Salts of the polyphosphate besides calcium can be used such as strontium, barium, manganese, magnesium, lithium, sodium, potassium, rubidium or cesium. Such metals are in Group IA and IIA of the periodic table.
The phospholipids are preferably:
(1) 1,palmitoyl-2,oleoylphosphatidylcholine (Avanti Polar Lipids, Birmingham, Ala.);
(2) E. coli phospholipids which are mainly phosphatidylethanolamine and phosphatidyl glycerol (4:1 mixture) with mixed fatty acyl chains mainly 16:0, 16:1, 18:1 (Avanti Polar Lipids). Many other synthetic and natural phospholipids can be used and other lipids such as triglycerides, cholesterol and the like can be added. These are described in Phospholipid Handbook, Marcel Dekker, Inc., New York 1-22 and 603-637 (1993).
The PHB and polyphosphate mixture can be dried by any means which does not decompose the PHB. A vacuum oven can be used for instance. A microwave oven can be used. PHB and polyPi can be mixed in a mortar and pestle, heated to the melting point of PHB (ca 175xc2x0 C.) and cooled slowly. The preferred ratio of PHB to CaPolyPi is between about 1 to 1 and 10 to 1 and most preferably 2:1 PHB to CaPolyPi in about 1% of phospholipid. The ratio of phospholipid to the mixture is between about 1000 to 1 and 100,000 to 1.
The membrane can be formed in any solvent in which the phospholipid, polyphosphate and PHB can be dissolved. Chloroform and dichloromethane have been used successfully. Mixing methods other than sonication can be used. Sonication is preferred.
Preferably, a solution of PHB in chloroform is added to dry pulverized Ca (PPi), the chloroform is evaporated and the mixture is microwaved to dry (4 min) Chloroform (dry) is added and the mixture is sonicated. The chloroform solution is added to the phospholipid in decane and the chloroform is evaporated to make a bilayer. If liposomes are made, phospholipid are added to the chloroform solution. The chloroform is evaporated, aqueous salt buffer is added to the dry film, and the mixture is sonicated.
The aqueous bathing solution for the membrane can be symmetric (same solution on both sides of membrane) or unsymmetric. Buffers are used to maintain the pH, preferably between about 5 and 9. The solutions preferably have a high ionic strength and contain an amount of magnesium salts.
The present invention is useful for assaying for calcium, or other metal, blocking compounds. Inorganic blocker compounds are for instance lanthanum, aluminum, nickel, cadmium, cobalt and manganese. Organic Ca2+ blockers are for instance nifedipine, verapamil and diltiazem.
The transport means can be ion concentration or voltage differentials. Preferably the aperture for the experiments with the voltage clamp has a cross-sectional area of between 50 and 1000 square xcexcm. The bilayer membrane has a thickness of between about 40 and 120 Angstroms.
Usually a bilayer membrane is formed. It will be appreciated that there can be multiple layers formed.